I. Plasminogen and Plasminogen Activators PA0 II. Inhibitors of Plasminogen Activators PA0 III. The Role of Plasminogen Activators in Cancer
The serum protein, plasminogen, plays an integral role in the proteolytic dissolution (or fibrinolysis) of blood clots. Plasminogen is an inactive "proenzyme." It has a specific affinity for fibrin, and thus becomes incorporated into blood clots as they form. Plasminogen's proteolytic activity is released by "plasminogen activators" ("PA") that specifically cleave the molecule to yield the active protease, plasmin. Plasmin is capable of digesting the fibrin threads of blood clots, as well as other substances involved in creating blood clots, such as fibrinogen, factor V, factor VIII, prothrombin, and factor XII (for review, see Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985), herein incorporated by reference)).
Plasmin is a serine protease, and exhibits substantial amino acid and mechanistic homology with trypsin, chymotrypsin, and pancreatic elastase. Plasmin has a relatively broad trypsin-like specificity, hydrolyzing proteins and peptides at lysyl and arginyl bonds (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dane, K. et al., Adv. Canc. Res. 44:139-266 (1985)).
Agents that are capable of activating plasminogen (i.e. converting it to plasmin) have been extensively studied. Two classes of natural mammalian plasminogen activators have been described: urokinase-type plasminogen activator and tissue-type plasminogen activator (Dane, K. et al., Adv. Canc. Res. 44:139-266 (1985); Devlin, et al., PCT appl. WO88/05081; Kasaia et al., U.S. Pat. No. 5,098,840; Hayashi, S. et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030; Hayashi, S. et al., U.S. Pat. No. 5,004,609; Pyke, C. et al., Amer. J. Pathol. 138:1059-1067 (1991); Madison, E. L. et al., Nature 339:721-724 (1989); Blasi, F. et al., J. Cell. Biol. 104:801-804 (1987)). These two classes of molecules can be distinguished immunologically, by tissue localization, and by the stimulation of their activity by fibrin. In addition, a third plasminogen activator, streptokinase, has also been described. Streptokinase differs from urokinase and tPA in that it is a bacterial protein produced by the streptococci.
Urokinase-type plasminogen activator (UK) is a multi-domain protein with one domain being a trypsin-like serine protease (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dane, K. et al., Adv. Canc. Res. 44:139-266 (1985); Stra.beta.burger, W. et al., FEBS Lett. 157:219-223 (1983)). This protease domain converts plasminogen to plasmin by cleavage at an arginyl residue (Castellino, R. W. et al., Meth. Enzymol. 80:365-380 (1981); Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). The amino acid sequence and three-dimensional structure of several serine proteases, including trypsin, chymotrypsin, and elastase have been deduced (Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985); Stra.beta.burger, W. et al., FEBS Lett. 157:219-223 (1983)).
Urokinase is synthesized in the kidneys, and can be recovered from urine. It is initially produced as a single chain protein, "pro-urokinase" that can be proteolytically cleaved by plasmin into an active two-chain protein (Devlin, et al., PCT appl. WO88/05081).
Tissue-type plasminogen activator (tPA) is produced by the cells that line the lumen of blood vessels or endothelial cells. Like urokinase, tPA is also initially produced as a single-chain molecule (Rijken, D. G. et al., J. Biol. Chem. 256:7035-7041 (1981); Pennica, D. et al., Nature 301:214-221 (1983)).
The known plasminogen activators differ significantly in characteristics such as their biological half-lives and their preference for fibrin. All three classes of activators have been widely used as thrombolytic agents for the treatment of thrombosis in myocardial infarction, stroke, arterial occlusion, etc. (Kasai et al., U.S. Pat. No. 5,098,840; Hayashi et al., U.S. Pat. No. 5,004,609; Hayashi et al., U.S. Pat. No. 4,851,345; Sasaki et al., U.S. Pat. No. 4,258,030).
The regulation of fibrinolysis is crucial to the normal functioning of the circulatory system. Thus, the activity of serum proteases (such as urokinase or tPA) capable of activating plasminogen must be carefully regulated to ensure that clot formation and dissolution can occur. One manner in which such control is mediated concerns the regulated synthesis of inhibitors of plasminogen activators.
Three classes of naturally occurring physiological inhibitors of the plasminogen activators have been identified: the endothelial cell type PA-inhibitor (PAI-1), the placental type PA-inhibitor (PAI-2), and protease nexin-I (Sprengers, E. D. et al., Blood 69:381-387 (1987); Blasi, F. et al., J. Cell Biol. 104:801-804 (1987); Madison, E. L. et al., Nature 339:721-724 (1989); Carrell, R. W. et al., Nature 353:576-578 (1991); Lostukoff et al., U.S. Pat. No. 4,952,512; all herein incorporated by reference). Such inhibitors comprise nearly 10% of the total protein in blood plasma. They control a variety of critical events associated with connective tissue turnover, coagulation, fibrinolysis, complement activation and inflammatory reactions. Their function in the regulation of the fibrinolytic system has not yet been fully clarified.
PAI-1 and PAI-2 have an approximate molecular weight of 50,000. They differ in immunological reactivity and in their physiological characteristics (Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)). Whereas these inhibitors are specific for PA, the protease nexin I inhibits plasmin, thrombin, and other trypsin-like serine proteases in addition to PA (Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)).
PAI-1 is synthesized by a wide variety of cell types, including endothelial cells, hepatocytes, several hepatoma cell lines, granulosa cells, and possibly smooth muscle cells (Sprengers, E. D. et al., Blood 69:381-387 (1987); Blasi, F. et al., J. Cell Biol. 104:801-804 (1987)). PAI-1 interacts with both urokinase and tPA to form a stable bimolecular complex that inactivates both the inhibitor and the PA (Kruithof, C.K.O. et al., Thromb.-Haemost. 55:65-68 (1986)). PAI-2 was first identified in placental tissue. The inhibitor has been purified to homogeneity and found to have a molecular weight of 48,000. It is not generally present in serum samples (Sprengers, E. D. et al., Blood 69:381-387 (1987)). Protease nexin I is a 43,000 protein that exhibits a broad activity against trypsin-like serine proteases. It is synthesized by fibroblasts, heart muscle cells, and kidney epithelial cells (Sprengers, E. D. et al., Blood 69:381-387 (1987)).
Metastasis involves the escape of a tumor cell from the tumor, its translation to a new site, and its successful invasion of the tissue of the new site and vascularization there to create a new tumor locus. The membranes of vascular or lymphatic vessels and dense connective tissue pose natural obstacles to the metastasis of tumor cells. The observation that explants of cancer tissue consistently caused proteolytic degradation eventually led to the recognition that tumor cells released a plasminogen activator capable of converting plasminogen to plasmin (see, for review, Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985)). By secreting such a plasminogen activator, tumor cells are able to initiate a cascade of reactions that results in the localized proteolysis needed for tumor cell dissemination (Ossowski, L., Cell 52:321-328 (1988); Yu, H. et al., Canc. Res. 50:7623-7633 (1990)).
Urokinase has been found to be secreted by a variety of tumor types, including lung, colon and breast (see, Dan.o slashed., K. et al., Adv. Canc. Res. 44:139-266 (1985) for review). Urokinase has been found to have an important role in the metastasis of tumor cells (Yu, H. et al., Canc. Res. 50:7623-7633 (1990); Ossowski, L., Cell 52:321-328 (1988); Ossoswki, L. et al., Canc. Res. 51:274-281 (1991)). A positive correlation has been reported between the metastatic potential of tumor cells and their capacity to produce urokinase (Axelrod, J. H. et al., Molec. Cell. Biol. 9:2133-2141 (1989); Yu, H. et al., Canc. Res. 50:7623-7633 (1990)).
Significantly, urokinase appears to mediate its effect at the initial stage of metastasis by facilitating the escape of tumor cells from the primary tumor site. Indeed, inhibitors of urokinase, tested in a metastatic mouse model, were ineffective in preventing either the translation or invasion of of metastatic cells that had been injected into the animal's bloodstream (Ostrowski, L. E. et al., Canc. Res. 46:4121-4128 (1986)). Thus, UK inhibitors would be an especially desirable inhibitor of tumor metastasis because they would not allow even the first stage of metastasis to occur (i.e. escape of cells from the primary tumor site).
As indicated, urokinase is also produced in response to many natural physiological conditions. The implantation of a fertilized egg into the uterine wall provides an example of localized proteolysis, mediated by urokinase, that occurs in normal tissue (Dane, K. et al., Adv. Canc. Res. 44:139-266 (1985)).
In view of the role of plasminogen activators, in general, and of urokinase, in particular, in the metastasis of tumor cells, and in mediating uterine implantation, it would be desirable to have a low molecular weight, reversible, proteinaceous inhibitor specific for urokinase that could be employed in the treatment of metastatic cancers, or in the prevention of pregnancy. No low molecular weight, protein inhibitors with significant functional affinity for UK have been previously identified. The present invention provides such molecules, and methods for their use.